Method for Preparing Porous Scaffold for Tissue Engineering, Cell Culture and Cell Delivery

ABSTRACT

The present invention relates to a method for preparing a porous scaffold for tissue engineering. It is another object of the present invention to provide a porous scaffold obtainable by the method as above described, and its use for tissue engineering, cell culture and cell delivery. The method of the invention comprises the steps consisting of: a) preparing an alkaline aqueous solution comprising an amount of at least one polysaccharide, an amount of a cross-linking agent and an amount of a porogen agent b) transforming the solution into a hydrogel by placing said solution at a temperature from about 4° C. to about 80° C. for a sufficient time to allow the cross-linking of said amount of polysaccharide and c) submerging said hydrogel into an aqueous solution d) washing the porous scaffold obtained at step c).

FIELD OF THE INVENTION

The present invention relates to a method for preparing a porousscaffold for tissue engineering. It is another object of the presentinvention to provide a porous scaffold obtainable by the method as abovedescribed, and its use for tissue engineering, cell culture and celldelivery

BACKGROUND OF THE INVENTION

Tissue engineering is generally defined as the creation of tissue ororgan equivalents by seeding of cells onto or into a scaffold suitablefor implantation. The scaffolds must be biocompatible and cells must beable to attach and proliferate on the scaffolds in order for them toform tissue or organ equivalents. These scaffolds may therefore beconsidered as substrates for cell growth either in vitro or in vivo.

The attributes of an ideal biocompatible scaffold would include theability to support cell growth either in vitro or in vivo, the abilityto support the growth of a wide variety of cell types or lineages, theability to be endowed with varying degrees of flexibility or rigidityrequired, the ability to have varying degrees of biodegradability, theability to be introduced into the intended site in vivo withoutprovoking secondary damage, and the ability to serve as a vehicle orreservoir for delivery of drugs or bioactive substances to the desiredsite of action.

A number of different scaffold materials have been utilized, for guidedtissue regeneration and/or as biocompatible surfaces. Biodegradablepolymeric materials are preferred in many cases since the scaffolddegrades over time and eventually the cell-scaffold structure isreplaced entirely by the cells. Among the many candidates that may serveas useful scaffolds claimed to support tissue growth or regeneration,are included gels, foams, sheets, and numerous porous particulatestructures of different forms and shapes.

Among the manifold natural polymers which have been disclosed to beuseful for tissue engineering or culture, one can enumerate variousconstituents of the extracellular matrix including fibronectin, varioustypes of collagen, and laminin, as well as keratin, fibrin andfibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate andothers.

Other common polymers that were used include poly(lactide-co-glycolide)(PLG). PLG are hydrolytically degradable polymers that are FDA approvedfor use in the body and mechanically strong (Thomson R C, Yaszemski M J,Powers J M, Mikos A G. Fabrication of biodegradable polymer scaffolds toengineer trabecular bone. J Biomater Sci Polym Ed. 1995; 7(1):23-38;Wong W H. Mooney D J. Synthesis and properties of biodegradable polymersused as synthetic matrices for tissue engineering. In: Atala A, Mooney DJ, editors; Langer R, Vacanti J P, associate editors. Syntheticbiodegradable polymer scaffolds. Boston: Birkhauser: 1997. p. 51-82).However, they are hydrophobic and typically processed under relativelysevere conditions, which make factor incorporation and entrapment ofviable cells potentially a challenge.

As an alternative, a variety of hydrogels, a class of highly hydratedpolymer materials (water content higher than 30% by weight), have beenused as scaffold materials. They are composed of hydrophilic polymerchains, which are either synthetic or natural in origin. The structuralintegrity of hydrogels depends on cross-links formed between polymerchains via various chemical bonds and physical interactions.

For example, document U.S. Pat. No. 6,586,246 B1 has disclosed a methodfor preparing a porous hydrogel scaffold which may be used as supportsfor tissue engineering or culture matrices. The method of the documentcomprises the steps consisting of a) dissolving a biodegradablesynthetic polymer in an organic solvent to prepare a polymeric solutionof high viscosity b) adding a porogen agent to this solution; c) castingthe polymer into a mould d) removing the organic solvent e) submergingthe organic solvent-free polymer/salt gel slurry in a hot aqueoussolution or acidic solution to cause the salt to effervesce at roomtemperature to form the porous scaffold. However, this method ofpreparation of a porous hydrogel involves the use of an organic solventwith a synthetic polymer which renders the method according to thisinvention weakly compatible with biological and therapeutic purposes.

Therefore there is still an existing need in the art to develop a methodfor preparing porous scaffold matrices that can be used for biologicaland therapeutic purposes.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a methodfor preparing a porous scaffold which comprises the steps consisting of:

-   -   a) preparing an alkaline aqueous solution comprising an amount        of at least one polysaccharide, an amount of a cross-linking        agent and an amount of a porogen agent.    -   b) transforming the solution into a hydrogel by placing said        solution at a temperature from about 4° C. to about 80° C. for a        sufficient time to allow the cross-linking of said amount of        polysaccharide and    -   c) submerging said hydrogel into an aqueous solution    -   d) washing the porous scaffold obtained at step c).

It is another object of the present invention to provide a porousscaffold obtainable by the method as above described.

It is still further an object of the present invention to provide theuse of porous scaffold of the invention for tissue engineering, cellculture and cell delivery.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “polysaccharide”, as used herein, refers to a moleculecomprising two or more monosaccharide units.

The term “alkaline solution”, as used herein, denotes a solution havinga pH superior to 7.

The term “acidic solution”, as used herein, denotes a solution having apH inferior to 7.

The term “aqueous solution”, as used herein, refers to a solution inwhich the solvent is water.

The term “cross-linking” refers to the linking of one polymer chain toanother one with covalent bonds.

The term “porogen agent” denotes any solid agent which has thecapability to form pores within a solid structure.

As used herein, a “scaffold” is defined as a semi-solid, systemcomprising a three-dimensional network of one or more species ofpolysaccharide chains. Depending on the properties of the polysaccharide(or polysaccharides) used, as well as on the nature and density of thenetwork, such structures in equilibrium can contain various amounts ofwater.

The term “cross-linking agent” includes any agent able to introducecross-link between the chains of the polysaccharides of the invention.

“Biodegradable”, as used herein, refers to materials that degrade invivo to non-toxic compounds, which can be excreted or furthermetabolized.

Porous Scaffolds and Method for Preparing Thereof

A first object of the invention relates to a method for preparing aporous scaffold which comprises the steps consisting of:

-   -   a) preparing an alkaline aqueous solution comprising an amount        of at least one polysaccharide, an amount of a covalent        cross-linking agent and an amount of a porogen agent    -   b) transforming the solution into a hydrogel by placing said        solution at a temperature from about 4° C. to about 80° C. for a        sufficient time to allow the cross-linking of said amount of        polysaccharide and    -   c) submerging said hydrogel into an aqueous solution    -   d) washing the porous scaffold obtained at step c).

In the present invention, any type of polysaccharide can be used.Synthetic or natural polysaccharides may be alternatively used for thepurpose of the invention. For example, suitable natural polysaccharidesinclude, but are not limited to, dextran, agar, alginic acid, hyaluronicacid, inulin, pullulan, heparin, fucoidan, chitosan, scleroglucan,curdlan, starch, cellulose and mixtures thereof. Monosaccharides thatmay be used to produce the desired polysaccharide include but are notlimited to ribose, glucose, mannose, galactose, fructose, sorbose,sorbitol, mannitol, iditol, dulcitol and mixtures thereof. Chemicallymodified polysaccharides bearing for instance acidic groups(carboxylate, sulphate, phosphate), amino groups (ethylene amine,diethylamine, diethylaminoethylamine, propylamine), hydrophobic groups(alkyl, benzyl) can be included. Many of these compounds are availablecommercially from companies such as Sigma-Aldrich (St. Louis, Mich.,US).

The preferred weight-average molecular weight for the polysaccharide isfrom about 10,000 Daltons to about 2,000,000 Daltons, more preferablyfrom about 10,000 Daltons to about 500,000 Daltons, most preferably fromabout 10,000 Daltons to about 200,000 Daltons.

In one embodiment of the invention, the polysaccharide(s) used toprepare the scaffold of the invention is a neutral polysaccharide suchas dextran, agar, pullulan, inulin, scleroglucan, curdlan, starch,cellulose or a mixture thereof. In a preferred embodiment, a mixture ofpullulan and dextran is used to prepare the scaffold of the invention.For example, said mixture comprises 25% of dextran and 75% of pullulan.

In another embodiment of the invention, the polysaccharide(s) used toprepare the scaffold of the invention is a positively chargedpolysaccharide such as chitosan, DEAE-dextran and mixtures thereof.

In another embodiment of the invention, the polysaccharide(s) used toprepare the scaffold of the invention is a negatively chargedpolysaccharide such as alginic acid, hyaluronic acid, heparin, fucoidanand mixtures thereof.

In another embodiment of the invention, the polysaccharide(s) used toprepare the scaffold of the invention is a mixture of neutral andnegatively charged polysaccharides, wherein the negatively chargedpolysaccharides represents 1 to 20%, preferably 5 to 10% of the mixture.

In a particular embodiment the covalent cross-linking agent is selectedfrom the group consisting of trisodium trimetaphosphate (STMP),phosphorus oxychloride (POCl₃), epichlorohydrin, formaldehydes,hydrosoluble carbodiimides, glutaraldehydes or any other compound thatis suitable for crosslinking a polysaccharide. In a preferredembodiment, the cross-linking agent is STMP. The concentration of thecovalent cross-linking agent in the aqueous solution (w/v) is from about1% to about 6%, more preferably from about 2% to about 6%, mostpreferably from about 2% to about 3%. It is preferred to use thecross-linking agent at such an amount that the weight ratio of thepolysaccharide to the cross-linking agent is in the range from 20:1 to1:1, preferably from 15:1 to 1:1 and more preferably from 10:1 to 1:1.

Many of these compounds are available commercially from companies suchas Sigma-Aldrich (St. Louis, Mich., US).

The aqueous solution comprising the polysaccharide may further comprisevarious additives depending on the intended application. Preferably, theadditive is compatible with the polysaccharide and does not interferewith the effective cross-linking of the polysaccharide(s). The amount ofthe additive used depends on the particular application and may bereadily determined by one skilled in the art using routineexperimentation.

The aqueous solution comprising the polysaccharide may optionallyinclude at least one antimicrobial agent. Suitable antimicrobialpreservatives are well known in the art. Examples of suitableantimicrobials include, but are not limited to, alkyl parabens, such asmethylparaben, ethylparaben, propylparaben, and butylparaben; cresol;chlorocresol; hydroquinone; sodium benzoate; potassium benzoate;triclosan and chlorhexidine. Other examples of antibacterial agents andof anti-infectious agents that may be used are, in a nonlimiting manner,rifampicin, minocycline, chlorhexidine, silver ion agents andsilver-based compositions.

The aqueous solution comprising the polysaccharide may also optionallyinclude at least one colorant to enhance the visibility of the solution.Suitable colorants include dyes, pigments, and natural coloring agents.Examples of suitable colorants include, but are not limited to, alcianblue, fluorescein isothiocyanate (FITC) and FITCdextran.

The aqueous solution comprising the polysaccharide may also optionallyinclude at least one surfactant. Surfactant, as used herein, refers to acompound that lowers the surface tension of water. The surfactant may bean ionic surfactant, such as sodium lauryl sulfate, or a neutralsurfactant, such as polyoxyethylene ethers, polyoxyethylene esters, andpolyoxyethylene sorbitan.

In a particular embodiment, the porogen agent may be an agent that canbe transformed into a gas in acidic conditions, with pores being formedby the carbon dioxide molecules that leach out from the polymer.Examples of such a porogen agent include but are not limited to ammoniumcarbonate, ammonium bicarbonate, sodium carbonate, and sodiumbicarbonate, calcium carbonate and mixtures thereof. It is preferred touse the porogen agent at such an amount that the weight ratio of thepolysaccharide to the porogen agent is in the range from 6:1 to 1:1,preferably from 4:1 to 1:1, more preferably to 2:1 to 1:1. Many of thesecompounds are available commercially from companies such asSigma-Aldrich (St. Louis, Mich., US). In one embodiment, the ratio ofthe polysaccharide to the porogen agent may be in the range from 6:1 to0.5:1, preferably from 4:1 to 0.5:1, more preferably to 2:1 to 0.5:1. Inanother embodiment, while the polysaccharide is a positively chargedpolysaccharide, the ratio of the polysaccharide to the porogen agent maybe in the range from 50:1 to 1:1, preferably from 20:1 to 1:1 and morepreferably from 10:1 to 1:1.

In this particular embodiment, the aqueous solution of step c) is anacidic solution. The acid may be selected from the group consisting ofcitric acid, hydrochloric acid, acetic acid, formic acid, tartaric acid,salicylic acid, benzoic acid, and glutamic acid.

Alternatively, the porogen agent may be an inorganic salt that can bedissolved once the cross-linked polysaccharide scaffold is immersed inwater. An example of such a porogen agent includes saturated saltsolution, which would be dissolved progressively. In this particularembodiment, the aqueous solution of step c) is an aqueous solution,preferably water, and more preferably distilled water.

The concentration of the porogen agent affects the size of the poresformed in the scaffolds, so that the pore size can be under the controlof the concentration of said porogen agent.

The average pore size of the scaffold is from about 1 μm to about 500μm, preferably from about 150 μm to about 350 μm, more preferably fromabout 175 μm to about 300 μm. The density of the pores or porosity isfrom about 4% to about 75%, preferably from about 4% to about 50%.

In another embodiment, the method of the invention may comprise afurther step consisting of freeze-drying the scaffold obtained at stepd). Freeze-drying may be performed with any apparatus known in the art.There are essentially three categories of freeze dryers: rotaryevaporators, manifold freeze dryers, and tray freeze dryers. Suchapparatus are well known in the art and are commercially available suchas a freeze-dryer Lyovac (GT2, STERIS Rotary vane pump, BOC EDWARDS).Basically, the vacuum of the chamber is from 0.1 mBar to about 6.5 mBar.The freeze-drying is performed for a sufficient time sufficient toremove at least 98.5% of the water, preferably at least 99% of thewater, more preferably at least 99.5%.

In another embodiment, the method of the invention may comprise afurther step consisting of hydrating the scaffold as prepared accordingto the invention.

Said hydration may be performed by submerging the scaffold in an aqueoussolution (e.g., de-ionized water, water filtered via reverse osmosis, asaline solution, or an aqueous solution containing a suitable activeingredient) for an amount of time sufficient to produce a scaffoldhaving the desired water content. For example, when a scaffoldcomprising the maximum water content is desired, the scaffold issubmerged in the aqueous solution for an amount of time sufficient toallow the scaffold to swell to its maximum size or volume. Typically,the scaffold is submerged in the aqueous solution for at least about 1hour, preferably at least about 2 hours, and more preferably about 4hours to about 24 hours. It is understood that the amount of timenecessary to hydrate the scaffold to the desired level will depend uponseveral factors, such as the composition of the used polysaccharides,the size (e.g., thickness) of the scaffold, and the temperature of theaqueous solution, as well as other factors.

In a particular embodiment, the hydrated scaffold comprises 80% ofwater, preferably 90% of water, most preferably 95% of water.

In another particular embodiment, the aqueous solution of step a) may bepoured in a mould before step b), so that the porous scaffold obtainedwith the method of the invention can take a desired form. Anygeometrical moulds may be used according to the invention. Differentsizes may be also envisaged. For example, typically, the aqueoussolution may be poured in a tubular mould with a central axis so thatthe porous scaffold may be tubular with a desired external and internaldiameter. The mould may be made of any material, but preferred materialincludes non sticky surfaces such as Teflon.

Alternatively, the scaffolds of the invention may be cut and shaped totake a desired size and form.

The methods of the invention can further include the step of sterilizingthe scaffold using any suitable process. The scaffold can be sterilizedat any suitable point, but preferably is sterilized before the scaffoldis hydrated. A suitable irradiative sterilization technique is forexample an irradiation with Cesium 137, 35 Gray for 10 minutes. Suitablenon-irradiative sterilization techniques include, but are not limitedto, UV-exposure, gas plasma or ethylene oxide methods known in the art.For example, the scaffold can be sterilized using a sterilisation systemwhich is available from Abtox, Inc of Mundelein, Ill. under the trademark PlazLyte, or in accordance with the gas plasma sterilizationprocesses disclosed in U.S. Pat. No. 5,413,760 and U.S. Pat. No.5,603,895.

The scaffold produced by the methods of the invention can be packaged inany suitable packaging material. Desirably, the packaging materialmaintains the sterility of the scaffold until the packaging material isbreached.

In another embodiment, one or more biomolecules may be incorporated inthe porous scaffold. The biomolecules may comprise, in otherembodiments, drugs, hormones, antibiotics, antimicrobial substances,dyes, radioactive substances, fluorescent substances, anti-bacterialsubstances, chemicals or agents, including any combinations thereof. Thesubstances may be used to enhance treatment effects, enhancevisualization, indicate proper orientation, resist infection, promotehealing, increase softness or any other desirable effect. In saidembodiment, the scaffold of the invention, comprising one or morebiomolecules as described here above, may be used as a controlledrelease system of an active agent.

The scaffold produced by the methods of the invention is free fromgrowth factors and other growth stimulants. In one embodiment, thebiomolecule may comprise chemotactic agents, antibiotics, steroidal ornon-steroidal analgesics, antiinflammatories, immunosuppressants,anti-cancer drugs, various proteins (e.g., short chain peptides, bonemorphogenic proteins, glycoprotein and lipoprotein); cell attachmentmediators; biologically active ligands; integrin binding sequence;ligands; various growth and/or differentiation agents (e.g., epidermalgrowth factor, IGF-I, IGF-II, TGF-[beta], growth and differentiationfactors, stromal derived factor SDF-1; vascular endothelial growthfactors, fibroblast growth factors, platelet derived growth factors,insulin derived growth factor and transforming growth factors,parathyroid hormone, parathyroid hormone related peptide, bFGF;TGF[beta] superfamily factors; BMP-2; BMP-4; BMP-6; BMP-12; sonichedgehog; GDF5; GDF6; GDF8; PDGF); small molecules that affect theupregulation of specific growth factors; tenascin-C; hyaluronic acid;chondroitin sulfate; fibronectin; decorin; thromboelastin;thrombin-derived peptides; heparin-binding domains; heparin; heparansulfate; DNA fragments, DNA plasmids, Si-RNA, transfection agents or anycombination thereof.

In one embodiment growth factors include heparin binding growth factor(HBGF), transforming growth factor alpha or beta (TGF.beta.), alphafibroblastic growth factor (FGF), epidermal growth factor (TGF),vascular endothelium growth factor (VEGF), and SDF-1, some of which arealso angiogenic factors. In another embodiment factors include hormonessuch as insulin, glucagon, and estrogen. In some embodiments it may bedesirable to incorporate factors such as nerve growth factor (NGF) ormuscle morphogenic factor (MMF). In one embodiment, TNF alpha/beta, orMatrix metalloproteinases (MMPs) are incorporated.

Additionally, scaffolds of the invention may optionally includeanti-inflammatory agents, such as indomethacin, salicylic acid acetate,ibuprofen, sulindac, piroxicam, and naproxen; thrombogenic agents, suchas thrombin, fibrinogen, homocysteine, and estramustine; andradio-opaque compounds, such as barium sulfate, gold particles and ironoxide nanoparticles (USPIOs).

Additionally, scaffolds of the invention may optionally compriseanti-thrombotic agents such as antivitamin K or aspirin, antiplateletagents such as aspirin, thienopyridine, dipyridamole or clopidogrel(that selectively and irreversibly inhibits adenosine diphosphate(ADP)-induced platelet aggregation) or anticoagulant agent such asheparin or fucoidan. The combination of heparin (anticoagulant) andtirofiban (antiplatelet agent) has been shown to be effective inreducing both thrombus and thromboemboli and may be incorporated.Genistein, a potential isoflavone which possesses dose-dependentantiplatelet and antiproliferative properties and inhibitscollagen-induced platelet aggregation responsible for primarythrombosis, may also be incorporated.

Methods for Using the Scaffolds of the Invention

Scaffolds of the invention are especially suited for tissue engineering,repair or regeneration. A difference in porosity may facilitatemigration of different cell types to the appropriate regions of thescaffold. In another embodiment, a difference in porosity may facilitatedevelopment of appropriate cell-to-cell connections among the cell typescomprising the scaffold, required for appropriate structuring of thedeveloping/repairing/regenerating tissue. For example, cell processesextension may be accommodated more appropriately via the varied porosityof the scaffolding material. Therefore, the scaffold may comprise cellsof any tissue.

In particular embodiment, the cells are seeded on said scaffold. Inanother embodiment, the scaffolds of the invention are submerged in aculture solution comprising the desired cells for an amount of timesufficient to enable penetration of the cells throughout the scaffold.

In another embodiment, scaffold of the invention is capable ofsupporting the viability and the growth of seeded cells in culture overlong periods of time without inducing differentiation.

In another embodiment, scaffold of the invention provides an environmentfor unstimulated cell growth (without activation by growth stimulants)

In another embodiment, scaffold of the invention can be used to studyphysiological and pathological processes such as tissue growth, boneremodeling, wound healing, tumorigenesis (including migration andinvasion), differentiation and angiogenesis. Scaffold allows thecreation of defined and controlled environments where specific processescan be modulated and studied in a controlled manner free of endogenousfactors.

In particular, scaffold of the invention can be used for 3D culture fordiagnostic or toxicological dosages. In this embodiment, the scaffold ofthe invention would allow evaluation of the toxicity of a productdirectly on cells present in a 3D environment. In said embodiment, thescaffold of the invention is used for cultivating cells useful for theevaluation of the toxicity and/or pharmacology of a product, such ashepatocytes, embryonic stem cells, epithelial cells, keratinocytes, orinduced pluripotent stem cells (iPS cells).

In another embodiment, scaffold of the invention is capable ofsupporting growth and differentiation of cell types in vitro and invivo.

In another embodiment, the cells are stem or progenitor cells. Inanother embodiment the cells may include but are not limited tochondrocytes; fibrochondrocytes; osteocytes; osteoblasts; osteoclasts;synoviocytes; bone marrow cells; mesenchymal cells; epithelial cells,hepatocytes, muscle cells; stromal cells; stem cells; embryonic stemcells; precursor cells derived from adipose tissue; peripheral bloodprogenitor cells; stem cells isolated from adult tissue; inducedpluripotent stem cells (iPS cells); genetically transformed cells; acombination of chondrocytes and other cells; a combination of osteocytesand other cells; a combination of synoviocytes and other cells; acombination of bone marrow cells and other cells; a combination ofmesenchymal cells and other cells; a combination of stromal cells andother cells; a combination of stem cells and other cells; a combinationof embryonic stem cells and other cells; a combination of progenitorcells isolated from adult tissue and other cells; a combination ofperipheral blood progenitor cells and other cells; a combination of stemcells isolated from adult tissue and other cells; and a combination ofgenetically transformed cells and other cells.

In another embodiment, any of these cells for use in the scaffolds andmethods of the invention, may be genetically engineered to express adesired molecule, such as for example green fluorescent protein (GFP),reporter gene (luciferase, phosphatise alkaline), heparin binding growthfactor (HBGF), transforming growth factor alpha or beta (TGF.beta.),alpha fibroblastic growth factor (FGF), epidermal growth factor (TGF),vascular endothelium growth factor (VEGF) and SDF-1, some of which arealso angiogenic factors. In another embodiment expressed factors includehormones such as insulin, glucagon, and estrogen. In another embodimentfactors such as nerve growth factor (NGF) or muscle morphogenic factor(MMF), or in another embodiment, TNF alpha/beta are expressed.

In a particular embodiment, scaffolds of the invention are suitable toprepare vascular substitutes to replace compromised arteries asdescribed for example, in Chaouat et al. (Chaouat M, Le Visage C,Autissier A, Chaubet F, Letourneur D. The evaluation of a small-diameterpolysaccharide-based arterial graft in rats. Biomaterials. 2006 Nov.;27(32):5546-53. Epub 2006 Jul. 20.). Such substitutes may be preparedaccording to the methods of the invention by using a mould as abovedescribed. Such substitutes may then comprise a population of cells toreconstruct in vitro or in vivo a vessel. In another embodiment thecells may include but are not limited to Mesenchymal Stem Cells (MSC),Endothelial Progenitor cells (EPCs), endothelial cells, fibroblasticcells and smooth muscle cells.

In another particular embodiment, scaffolds of the invention aresuitable to prepare cartilage or bone implants. In such a way, thescaffolds of the invention may be loaded with chondrocytes, osteocytes;osteoblasts; osteoclasts; vascular cells or mixtures thereof, and may becultured in presence of differentiating agents.

The site of implantation is dependent on the diseased/injured tissuethat requires treatment. For example, to treat structural defects inarticular cartilage, meniscus, and bone, the cell-seeded compositescaffold will be placed at the defect site to promote repair of thedamaged tissue.

In case of central nervous system (CNS) injuries, the composite scaffoldcan be seeded with a combination of adult neuronal stem cells, embryonicstem cells, glial cells and Sertoli cells. In the preferred embodiment,the composite scaffold can be seeded with Sertoli cells derived fromtransformed cell lines, xenogeneic or allogeneic sources in combinationwith neuronal stem cells. The Sertoli cells can be cultured with thecomposite scaffold for a period before addition of stem cells andsubsequent implantation at the site of injury. This approach cancircumvent one of the major hurdles of cell therapy for CNSapplications, namely the survival of the stem cells followingtransplantation. A composite scaffold that entraps a large number ofSertoli cells can provide an environment that is more amenable for thesurvival of stem cells.

Accordingly, the porous polysaccharide scaffold, which is preparedaccording to the present invention, can be effectively used as a rawmaterial for fabricating artificial tissues or organs such as artificialblood vessels, artificial bladder, artificial esophagus, artificialnerves, artificial hearts, prostatic heart valves, artificial skins,orthopedic implants, artificial muscles, artificial ligaments,artificial respiratory organs, etc. Further, the porous polysaccharidescaffold of the present invention can be prepared in the form of ahybrid tissue by blending or incorporating on or into other types ofbiomaterials and with functional cells derived from tissues or organs.It may have various biomedical applications, for example, to maintaincell functions, tissue regeneration, etc.

Alternatively scaffolds of the invention may be used for cell delivery.Actually, scaffolds of the invention may be used as a raw material forpreparing cell delivery systems that can be administered to a subjectfor therapeutic or diagnostic purposes. In a particular embodiment,scaffolds of the invention may be used to prepare a patch, a biofilm ora dressing that can be loaded with cells. For example, scaffolds of theinvention may used to prepare a dressing that can be applied on theskin, for reconstructing or healing the skin. Alternatively, saiddressing may used to be applied on the heart of a subject for treatingischemia (myocardial infarction). In those embodiments, the cells thatare entrapped in the scaffold can thus migrate into the targeted tissueor organ.

In another embodiment, scaffolds of the invention may be used forculturing cells. Cells may then be stimulated to undergo growth ofdifferentiation or other physiological processes by the addition ofappropriate growth factors. Culture medium containing one or morecytokines, growth factors, hormones or a combination thereof, may beused for maintaining cells in an undifferentiated state, or fordifferentiating cells into a particular pathway.

More particularly, the scaffold of the invention may be used forproducing molecules of interest. Actually, scaffolds of the inventionmay be used to provide a biological environment for the anchorage ofcells in a bioreactor, so that the cells can produced the desiredmolecules. The scaffolds of the invention provide mechanical andbiochemical protection of the cultured cells.

The scaffolds may thus serve as a cell reservoir for producing desiredmolecules such as proteins, organic molecules, and nucleotides. Forexample, proteins of interest include but are not limited to growthfactors, hormones, signal molecules, inhibitors of cell growth, andantibodies. Scaffolds of the invention are particularly interesting forproducing monoclonal antibodies. Scaffolds of the invention may be alsosuitable to produce organic molecules such as flavours, therapeuticmolecules . . . .

In this purpose, the scaffolds of the invention may be loaded with anytype of cells, including prokaryotic and eukaryotic cells. For examples,scaffolds of the invention may be load with bacteria, yeast cells,mammalian cells, insect cells, plant cells, etc. Specific examplesinclude E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian celllines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as wellas primary or established mammalian cell cultures (e.g., produced fromlymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervouscells, adipocytes, etc.). More particularly, the invention contemplatesthe use of established cell lines such as hybridomas. Alternatively, thecells may be genetically engineered to express a desired molecule asdescribed above.

The scaffold of the invention may be loaded with cells, cultured for acertain period of time then the cells can beretrieved/extracted/separated from the scaffold for further use, such astherapeutic or diagnostic applications or cell analysis. Separation ofthe cells from the scaffold may involve the use of enzymes that coulddegrade the scaffold, such as pullulanase and/or the use of enzymes thatcould detach the cells such as collagenase, elastase, trypsin orcell-detaching solutions such as EDTA.

The invention will further be illustrated in view of the followingfigures and examples.

FIGURES

FIG. 1: A porous scaffold obtained as in Example 1 (Scale: 6 mm)

FIG. 2: A porous scaffold obtained as in Example 1: scanning ElectronMicroscopy analysis of the scaffold (right image, scale: 200 microns).

FIG. 3: Formazan absorbance (570 nm) at day 1 as a function of theinitial number of cells seeded on porous scaffolds.

EXAMPLES Example 1 Polysaccharides-Based Scaffolds Preparation

Polysaccharide-based scaffolds were prepared using a mixture ofpullulan/dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc., Okayama,Japan; dextran MW 500,000, Pharmacia). A polysaccharide solution wasprepared by dissolving 9 gr of pullulan and 3 gr of dextran into 40 mLof distilled water. Sodium carbonate (8 g) was then added to thepolysaccharide solution and stirring was maintained until a homogeneousmixture was obtained. Chemical cross-linking of polysaccharide wascarried out using the cross-linking agent trisodium trimetaphosphateSTMP (Sigma, St Louis) under alkaline condition. Briefly, one milliliterof 10M sodium hydroxide was added to 10 g of the polysaccharidesolution, followed by the addition of one milliliter of water containing300 mg of STMP. The mixture was then poured into petri dishes (Nunclon®,#150288) and incubated at 50° C. for 15 min. Resulting hydrogels wereimmediately immersed into a large beaker containing a 20% acetic acidsolution, for at least 30 minutes. Resulting scaffolds were washedextensively with phosphate buffer saline pH 7.4 then with distilledwater for at least 2 days. After a freeze-drying step, porous scaffoldswere stored at room temperature until use. Scanning Electron Microscopyanalysis confirmed the porosity of the scaffolds (FIGS. 1 and 2).

Example 2 Types of Polysaccharides

Porous scaffolds were prepared as described in example 1, usingdifferent types and ratios of polysaccharides, while keeping the totalamount of polysaccharide at a constant value. Polysaccharides wereeither pullulan, dextran 500, fucoidan LMW (Low Molecular Weight) andfucoidan HMW (High Molecular Weight).

Dextran Fucoidan Fucoidan Pullulan 500 LMW HMW Solubilization Viscosity100%  +++ +++ 100%  +/− + 50% 50% ++ ++ 75% 25% ++ ++ 75% 25% +/− +++75% 25% + +

Solubilization (+++ indicates a complete solubilization of thepolysaccharides) and viscosity of the resulting polysaccharide solution(+++ indicates a very high viscosity of the solution) were visuallyassessed. In all cases, porous scaffolds were obtained at the end of theprotocol.

Example 3 Porogen Amount

Porous scaffolds were prepared as described in example 1, while varyingthe amount of the porogen agent. Briefly, 2, 4 or 8 gr of sodiumcarbonate were added to the pullulan/dextran solution.

Porogen agent Solubilization Viscosity Porosity 2 g ++ ++ + 4 g ++ ++ ++8 g ++ ++ ++

Solubilization (++ indicates a complete solubilization of thepolysaccharides), viscosity of the resulting polysaccharide solution(+++ indicates that a very high viscosity of the solution) and porositywere visually assessed. For scaffolds prepared with the lowest amount ofporogen (2 g), the effervescence process was moderate, as compared tothe effervescence obtained with 4 g and 8 g of porogen agent. In allcases, porous scaffolds were obtained at the end of the protocol.

Example 4 Cross-Linker Concentration

Porous scaffolds were prepared as described in example 1, while varyingthe amount of the cross-linking agent from 200 mg to 500 mg.

Cross-linking agent Solubilization Viscosity Porosity 200 mg ++ ++ ++300 mg ++ ++ ++ 400 mg ++ +++ ++ 500 mg ++ +++ +

Solubilization (+++ indicates a complete solubilization of thepolysaccharides), viscosity of the resulting polysaccharide solution(+++ indicates that a very high viscosity of the solution) and porositywere visually assessed. In all cases, porous scaffolds were obtained atthe end of the protocol.

Example 5 Cell Loading into the Porous Scaffolds

Human bone marrow Mesenchymal Stem Cells (hMSC) were cultured onscaffolds prepared as in Example 1. A circular punch was used to cut 6mm diameter and 1 mm thickness round-shaped porous scaffolds. Culturemedium consisted of low glucose DMEM (Gibco, Life Technology, New York)with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma).After cell trypsinization, rehydration of the dried scaffold wasperformed with 20 μL of cell suspension (10⁶ cells/scaffold). Sampleswere then maintained in 1 mL of culture medium for up to 1 week.Non-seeded porous scaffolds incubated in culture medium were used ascontrols.

A metabolic assay (MTT, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was performed to assess the cell viability.Briefly, a 5 mg/mL stock solution of MU (Sigma) was mixed 1:10 withDMEM. Scaffolds were incubated for 3 h at 37C with 1 mL of the reagentsolution. After washing the scaffolds with PBS, the formazan crystalswere solubilized in 0.3 mL of Isopropranol/HCl 0.04M. Absorbance wasrecorded at 590 nm with a microplate reader (Multiskan, Thermo ElectronCorporation, Waltham, Mass.). Absorbance at day 1 was directlyproportional to the initial number of cells seeded in the scaffolds(FIG. 3).

Similar experiments were successfully carried out with other cell typessuch as primary vascular smooth muscle cells and endothelial cells fromanimal and human origin.

Example 6 Confocal Analysis of Cell Behavior within the Porous Scaffolds

Fluorescent scaffolds were prepared as in example 1, by adding a smallamount (5 mg) of FITC-dextran to the polysaccharide solution.Fluorescent scaffolds were seeded as in Example 5, with hMSC labeledwith a fluorescent marker (PKH26, SIGMA P9691) according to themanufacturer's instructions). Confocal imaging confirmed the porousstructure of the scaffold.

Example 7 Cell Viability by Live and Dead Assay

Confocal imaging was used to assess the cell viability with a live/deadassay (Calbiochem, San Diego, Calif.), based on the use of twofluorescent probes that measure the cell membrane permeability: acell-permeable green fluorescent dye to stain live cells (calcein AM)and a cell nonpermeable red fluorescent dye (propidium iodide) to staindead cells. At day 7, most of the cells were live cells, with only fewdead cells found within the scaffolds.

Example 8 Influence of the Porogen Agent on Scaffold Porosity

Porous scaffolds were prepared as described in example 1, while varyingthe amount and the nature of the porogen agent. For confocal analysis offluorescent porous scaffolds, 5 mg of FITC-dextran were added to thepolysaccharide solution. Optical sections were acquired using a ZeissLSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany), equippedwith a 10× Plan-NeoFluar objective lens (numerical aperture of 0.3)(Carl Zeiss). FITC-dextran was excited at 488 nm with an argon laser andits fluorescent emission was selected by a 505-530 nm bandpass filter.Pore size was assessed with ImageJ® software. Void volume was calculatedwith a statistics/volume measurement module from Amira® software andresults are expressed as a percentage of the scaffold volume.

Mean Void diameter volume Polysaccharides Porogen agent (μm) (%)Pullulan (9g) + Sodium Carbonate 195 37% dextran 500 (3g) (8 g) Pullulan(9g) + Sodium Carbonate 207 71% dextran 500 (3g) (8 g) + Sodium Chloride(2 g) Pullulan (9g) + Sodium Carbonate 272 59% dextran 500 (3g) (8 g) +Sodium Chloride (8 g)

Example 9 Positively Charged Polysaccharide

Positively charged porous scaffolds were prepared using DEAE-Dextran asthe only polysaccharide. Briefly, DEAE-dextran solution was prepared bydissolving 1 g of DEAE-dextran (Fluka reference #30461) into 1.5 mL ofdistilled water. Sodium carbonate (100 mg) was then added to thepolysaccharide solution and stirring was maintained until a homogeneousmixture was obtained. Chemical cross-linking of polysaccharide wascarried out using the cross-linking agent trisodium trimetaphosphateSTMP (Sigma, St Louis) under alkaline condition. Briefly, 150 μL of 10Msodium hydroxide was added to the polysaccharide solution, followed bythe addition of 150 μL of water containing 45 mg of STMP. The mixturewas then poured into petri dishes (Nunclon®, #150288) and incubated at50° C. for 15 min. Resulting hydrogels were immediately immersed into alarge beaker containing a 20% acetic acid solution, for at least 30minutes. Resulting scaffolds were washed extensively with phosphatebuffer saline pH 7.4 then with distilled water for at least 2 days.After a freeze-drying step, porous scaffolds were obtained and stored atroom temperature until use.

Example 10 Negatively Charged Polysaccharide

Negatively charged porous scaffolds were prepared by adding fucoidan(Sigma reference #F5631) to a pullulan/dextran mixture. Briefly, apolysaccharide solution was prepared by dissolving 9 g of pullulan and 3g of dextran into 40 mL of distilled water, then adding 1.2 g offucoidan into the polysaccharide solution. Sodium carbonate (8 g) wasthen added to the polysaccharide solution and the cross-linking processwas carried out as described in Example 1 to obtain a 3D scaffold thatcontains a negatively charged polysaccharide.

Example 11 Differentiation of Human Mesenchymal Stem Cells intoChondrocyte-Like Cells in 3D Scaffolds

Human bone marrow Mesenchymal Stem Cells (hMSC) were cultured onscaffolds prepared as in Example 1 in serum-free chondrogenic medium.Chondrogenic medium consisted of DMEM supplemented with 10 ng/ml TGF-β3(Oncogene, Cambridge, Mass.), 100 nM dexamethasone (Sigma, St Louis,Mo.), 170 μM ascorbic acid 2-phosphate (Sigma, St Louis, Mo.) and 5 mLof ITS-plus (Collaborative Biomedical Products, Bedford, Mass.). After 3weeks of culture, seeded scaffolds were fixed in formaldehyde 10% thencryosectioned. Frozen sections were stained with either 0.05% (w/v)toluidine blue or with 0.1% safranin O solution. A strong positivestaining for extracellular matrix synthesis was observed, indicating MSCdifferentiation into cartilage cells.

Example 12 3D Culture of Hepatocytes

HepG2 cells, human hepatocellular carcinoma cells, were cultured in lowglucose DMEM (Gibco, Life Technology, New York, USA) with 10% fetalbovine serum and 1% penicillin/streptomycin (Sigma) on scaffoldsprepared as in Example 1. A circular punch was used to cut 6 mm diameterand 1 mm thickness round-shaped porous scaffolds.

After cell trypsinization, rehydration of the dried scaffold wasperformed with 20 μL of cell suspension (85,000 cells/scaffold). Sampleswere then maintained in 1 mL of culture medium for up to 1 week.Non-seeded porous scaffolds incubated in culture medium were used ascontrols. Hepatocyte spheroids formation was observed after 4 days ofculture. Cell viability in spheroids was assayed using Calcein AM(Calbiochem, San Diego Calif., USA) which is a polyanionic dyehydrolyzed by live cells thus producing an intense uniform greenfluorescence (wavelength 485-535 nm), according to the manufacturer'sinstructions. The seeded scaffolds contained living hepatocytes suitablefor pharmaco-toxicological assays.

1.-19. (canceled)
 20. A porous scaffold obtainable by a methodcomprising the following steps: a) preparing an alkaline aqueoussolution comprising an amount of at least one polysaccharide selectedfrom the group consisting of dextran, agar, pullulan, inulin, heparin,fucoidan, and mixtures thereof; an amount of a covalent cross-linkingagent selected from the group consisting of trisodium trimetaphosphate(STMP), phosphorus oxychloride (POCl₃), epichlorohydrin, formaldehydes,and glutaraldehydes; and an amount of a porogen agent selected from thegroup consisting of ammonium carbonate, ammonium bicarbonate, calciumcarbonate, sodium carbonate, and sodium bicarbonate and mixturesthereof; b) transforming the solution into a hydrogel by placing saidsolution at a temperature from about 4° C. to about 80° C. for asufficient time to allow the cross-linking of said amount ofpolysaccharide; c) submerging said hydrogel into an aqueous acidicsolution to form pores; and d) washing the porous scaffold obtained atstep c), wherein the porous scaffold is a hydrogel; the porogen agent isdissolved in step a); the weight ratio of the polysaccharide to theporogen agent is in the range from 6:1 to 1:1; and wherein the alkalineaqueous solution in a) does not include a surfactant.
 21. The porousscaffold according to claim 20 wherein the weight ratio of thepolysaccharide to the cross-linking agent is in the range from 15:1 to1:1.
 22. The porous scaffold according to claim 20 wherein the solutionof step a) is poured in a mould before step b).
 23. The porous scaffoldaccording to claim 20 wherein said scaffold is shaped.
 24. The porousscaffold according to claim 20 wherein the porogen agent used in step a)is selected from the group consisting of ammonium carbonate, calciumcarbonate, sodium carbonate, sodium bicarbonate, and mixtures thereof.25. The porous scaffold according to claim 20 wherein the size of thepores is comprised between 1 μm and 500 μm.
 26. The porous scaffoldaccording to claim 20 wherein the porosity is in the range from 4% to50%.
 27. The porous scaffold according to claim 20 loaded with an amountof cells.
 28. The porous scaffold according to claim 27 wherein thecells are selected in the group consisting of yeast cells, mammaliancells, insect cells, and plant cells.
 29. The porous scaffold accordingto claim 28 wherein mammalian cells are selected from the groupconsisting of chondrocytes; fibrochondrocytes; osteocytes; osteoblasts;osteoclasts; synoviocytes; bone marrow cells; epithelial cells,hepatocytes, mesenchymal cells; stromal cells; muscle cells, stem cells;embryonic stem cells; precursor cells derived from adipose tissue;peripheral blood progenitor cells; stem cells isolated from adulttissue; and genetically transformed cells.
 30. The porous scaffoldaccording to claim 20 for tissue engineering, cell culture and celldelivery.
 31. A vascular substitute made with a scaffold of claim 20.32. Cartilage or bone implants made with a scaffold of claim
 20. 33. Amethod for evaluating the toxicity and/or pharmacology of a productcomprising the use of a scaffold of claim
 20. 34. A controlled releasesystem of an active agent made with a scaffold of claim 20.